PY4302 Developmental Neuroscience Eye Development

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PY4302 Developmental NeuroscienceEye Development

• J. Martin Collinson
• School of Medical Sciences

m.collinson_at_abdn.ac.uk F 55750
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Vertebrate eye development Development of
the retina 2D patterning. Specification of
different cells. How does an apparently uniform
sheet of neural precursor cells differentiate
into a functional neural network with many
different neuronal cell types. Conservation of
genetic pathways controlling eye
development. Are vertebrate and invertebrate
eyes as different as they look?
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THE VERTEBRATE EYE

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Eye Development
Tissue sections through the head of a vertebrate
embryo
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Inductive interactions development of the lens.
The lens develops from and epithelial thickening
(placode) that forms in the surface of the head
after contact from underneath by the optic
vesicle.
Experiments by Spemann (1900-20s) and Lewis
(1904) suggested that contact by the optic
vesicle induced the formation of a lens placode
and was sufficient to force lens placode
formation in epithelia that would not normally
form the lens. THIS IS INDUCTION - CELLS THAT
WOULD BECOME SKIN HAVE BEEN TURNED INTO LENS AS A
RESULT OF CONTACT WITH OPTIC VESICLE http//zygot
e.swarthmore.edu/regul2.html
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Inductive interactions leading to lens
development Best data from Xenopus, may not be
same in other vertebrates (even amphibia).
Signals from dorsal mesoderm and from the gut
endoderm creates zone of lens competence in to
prospective head ectoderm. Signal from mesoderm
underlying prospective lens and planar signals
from developing neurectoderm further push
lens-competent epithelium to be
lens-biased. Signal from optic vesicle causes
formation of lens-determined placode. Once
tissue is determined it can only form lens. The
epithelial tissue is said to be lens-specified if
it will differentiate into lens-like bodies if
taken into culture (I.e needs no other signals to
complete full differentiation process).
See Grainger R. M. (1992). Embryonic lens
induction shedding light on vertebrate tissue
determination. Trends Genet. 8, 349-355.
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OV signal
Lens-determined
Lens-biased
Lens-specified
Lens-competent
The Valley of Lens Differentiation
Surface epithelium of head
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Molecular mediation of lens development 1 lens
competence
The transcription factor Pax6 is possibly
required for maintenance of lens competence in
mice. Expressed from early stages in head
ectoderm in a region which overlaps, but is wider
than the area fated to form the lens. In
chimeric mice that were a mixture of Pax6/ and
Pax6-/- cells, the mutant cells were excluded
from this wider area of the head ectoderm prior
to lens placode formation. I.e. need Pax6 to be
able to contribute to area of lens competence.
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Molecular mediation of lens development 2 lens
induction by the optic vesicle.
Molecules that secreted by the optic vesicle
function to induce lens development in competent
facial epithelium. Bone morphogenetic proteins
4 and 7 - Bmp4, Bmp7. (Evidence from
KOs). Fibroblast growth factors (?Fgf15)
(Evidence from dnFGFRs in lens placode). Something
else? (Bmps and Fgfs not enough in
culture). Their receptors are transmembrane
receptor tyrosine kinases or serine-threonine
kinases that set off secondary messenger pathways
in the prospective lens. These kick off the
genetic pathways that lead to lens development.
Fgf
Bmp
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Molecular mediation of lens development 3 lens
invagination and differentiation.
After induction of the lens placode, a genetic
pathway starts in the lens, marked by strong,
autoregulated expression of Pax6, and expression
of another transcription factor, Sox2. Sox2
complexes with Pax6 to control the expression of
other genes important for lens development.
Arm-waving model (not exact)
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DEVELOPMENT OF THE RETINA
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The adult retina in section
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Complex, but develops from a uniform sheet of
brain epithelium, early in development
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Anterior neural plate
Optic pits
Genetic patterning of the anterior normal plate
precedes the first signs of morphological
differentiation (the optic pits).
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WHY DO WE HAVE TWO EYES?
Many of the genes that will be used in retinal
development are already expressed across the
anterior neural plate at earliest
stages. Includes Pax6, Rx. These genes may be
turned on by signalling factors (Wnts) prior to
and during gastrulation.
Uniform early expression of Pax6 and Rx across
the anterior normal plate is split by the action
of a TGF-b type molecule called cyclops
(experiments in zebrafish). Action of cyclops is
possibly mediated via control of expression of
vertebrate hedgehog homologues (e.g. Sonic
hedgehog) that split the eye field, activate
expression of genes required to make optic stalk
(Pax2) and repress genes which are required for
retina (Pax6 and Rx).
cyc-/- or Shh-/- give cyclopic embryos.
Overexpression of Shh reduces the eye field.
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Pax6, Rx
cyc
Shh
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Shh
Pax2
Pax6
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Proximo-distal specification of the optic vesicle
The part of the optic vesicle in contact with the
head surface epithelium is termed distal and will
form the neural retina. Those proximal regions
become retinal pigment epithelium (RPE). How?
P
D
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Proximo-distal specification of the optic cup
Extrinsic signals from the overlying (lens
placode) epithelium turn on genes such as Chx10
and Lhx2 distally which are important for
development of neural retina.
Dissect off epithelium
No expression of distal genes
Expression of Chx10, Lhx2
Dissect out OV. Flip along P/D axis
P
D
P
D
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Morphological and cellular differentiation of the
retina
Mouse E10.5 optic cup
Undifferentiated neural retina
Optic stalk
Prospective retinal pigment epithelium (RPE)
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Proximo-distal specification of the optic cup
Gene knockouts (in vivo), and application of
growth factors in vitro all suggest that
fibroblast growth factors, FGFs, upregulate
neural retina genes and downregulate RPE genes.
Extra-ocular mesenchyme (the cells surrounding
the optic cup), upregulates genes specific for
the RPE.
Neural retina signals
RPE signals
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Differentiation of retinal cells
E10.5 mouse embryo - neural retina is composed of
a field of undifferentiated retinal progenitor
cells (RPCs). All RPCs express a common suite of
transcription factors. Pax6, Rx1, Six3, Six6,
Lhx2, Hes1. They are multipotent and can
differentiate into ganglion cells, bipolar,
amacrine, horizontal cells, photoreceptors and
Müller glia.
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The process of neuronal differentiation
begins. A wave of Sonic hedgehog (Shh)
expression sweeps through the retina. 1)
Propagates itself (Shh signals to cells ahead to
express more Shh). 2) Signals differentiation of
retinal ganglion cells (first retinal cells to
differentiate). Works via upregulation of a
transcription factor, Math5, in RGCs
Neumann Neusslein-Volhard. 2000. Science 289,
2137-2139
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Retinal cells differentiate in waves
Wang et al., 2002. Methods 28, 448-456.
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The undifferentiated neural retina consists of
multipotent retinal progenitor cells
Different retinal cell types are generated from
these progenitors in fixed chronological sequence
during late embryogenesis and early postnatal
life. Retinal ganglion cells and horizontal
cells are generated first, followed by cones,
amacrine cells, rods, bipolar cells and Müller
glia. (There is some overlap).
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The schedule of differentiation of retinal
progenitor cells (RPCs) is controlled by both
intrinsic genetic programs (not all RPCs are
equal) and extrinsic cues from their
environment. Cepko, C. L. (1999). The role of
intrinsic and extrinsic cues and bHLH genes in
the determination of retinal cell fates. Curr.
Opin. Neurobiol. 9, 37-46.
While Shh is sweeping through the ganglion cell
layer, there is an independent wave of Shh
sweeping through the inner nuclear layer, making
other neuronal cell types start to differentiate.
(2004. Development 131, 3849-3858)
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Intrinsic factors Some RPCs are biased to
produce certain types of progeny. E.g. some
RPCs (labeled with VC1.1 antisera) tend to
produce amacrine and horizontal cells.
Extrinsic factors Signals released by
differentiating or differentiated cells influence
the RPCs around them. E.g. amacrine cells
release a signal that inhibits further
differentiation of RPCs into amacrine cells in
culture. Limits numbers of amacrine cells
produced. Amacrine cells secret Shh, which acts
as a short range signal to direct differentiation
of other neurones. Similar results for ganglion
cells in chick retina.
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Arm-waving model
X
X
Intrinsic genetic programme is not locked -
influenced by environment and stochastic
process. RPCs can be bumped about experimentally
(I.e. persuaded to differentiate abnormally) but
maybe cant skip stages and cant go back.
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Genetic control of retinal neuron development
Stop proliferation
Neurogenic genes Proneural genes
The ciliary margin zone a domain of
undifferentiated retinal cells that can persist
into adulthood Genes expressed for first time in
red. Genes turned off in grey 1 stem cells. 2,
3 proliferating RPCs with evolving genetic
programme. 4 non-proliferating.
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Transcription factor control of retinal cell
specification
Two classes of transcription factor basic
helix-loop-helix (bHLH) TFs, and homeodomain
TFs. Expressed in overlapping but different
domains of the laminating retina. Work in
combination to modulate retinal progenitor fate.
Wang et al., 2005. Dev. Biol. 285, 101-115.
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e.g. these combinations of transcription factors
push RPCs into ganglion cell (GC) fate (red).
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rod
bipolar
Müller glia
ganglion cell
amacrine
Pax6 inactivated in RPCs
X
RPC
X
X
X
X
X
NeuroD
horiz.
rod
bipolar
Müller glia
ganglion cell
amacrine
Marquardt et al. (2001) Cell 105, 43-55.
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Prox1 also controls retinal development
RPC
RPC
NeuroD
Math5 Brn3b
X
horiz.
cone
rod
bipolar
Müller glia
ganglion cell
amacrine
Prox1 is a transcription factor Expression seems
to induce differentiation of RPCs Loss of Prox1
leads to loss of early-determined RPCs (espec.
horizontal cells) because they dont get the Prox
signal to differentiate. RPCs continue until they
get later rod/bipolar differentiation
signal. I.e. loss of Prox1 causes conversion one
sort of retinal cell into others. Remember this
for later.
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Dorso-ventral and naso-temporal specification of
the retina
D
T
N
V
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Retinal ganglion axons originating from specific
points along the nasotemporal or dorsoventral
axes of the retina stereotypically project to
specific points within the lateral geniculate
nucleus (LGN) and superior colliculus (SC). The
point-to-point topography of the retina is
therefore projected faithfully into the primary
visual centres in the mesencephalon and
diencephalon. How is this organised?
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Sperry, R. W. (1963). Proc. Natl. Acad. Sci.
(USA) 50, 703-710. The topographic targeting of
retinal ganglion cell (RGC) axons is governed by
graded distributions of molecules in the retina
and the tectum ( superior colliculus) that
confer positional addresses.
Now thought that these are chemical gradients of
a family of 14 transmembrane receptor tyrosine
kinases (Eph-receptors) in the retina and
topographically related gradients of their
ligands (ephrins) in the tectum/superior
colliculus.
Binding of an ephrin ligand to its Eph receptor
in a growth cone results in axonal repulsion.
(Mainly)
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In the developing retina, different Ephs are
localised in dorso-ventral or naso-temporal
gradients. Each retinal ganglion cell is
therefore uniquely labelled (painted) by the
levels of Ephs on its cell surface (e.g. has x
and y coordinates).
Potential targets of the RGC axons in the optic
tectum or superior colliculus are similarly
labelled by graded A/P and D/V gradients of
different ephrins.
Axons of RGCs will therefore project to the point
where they experience least repulsion (or most
attraction) in a pattern that faithfully
recapitulates the spatial origin of the RGCs in
the retina. Not necessarily absolutely true -
lots still to be discovered see OLeary, D. D. M
Wilkinson, D. G. (1999) Eph receptors and
ephrins in neural development. Curr. Opin.
Neurobiol. 9, 65-73.
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Retina
Tectum
T
P
A
N
Ephrin-A2
EphA3
D
D
V
V
EphB2/B3
Ephrin-B1 ??
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Membrane stripe assay of chemorepulsion Nasal
(left) and temporal (right) RGC axons grown in
culture and allowed to project over stripes (red)
of cell membranes of Cos cells transfected with
ephrin-A2 (A) or ephrin A5 (B,C). The red stripes
in B have a higher concentration of ephrin-A5
that those in C. A. Assume temporal RGCs are
expressing Eph receptors that are repulsed by
binding ephrin-A2, so their axons cannot grow
over transfected cells (so respect stripe
boundaries). Nasal axons not expressing these
Ephs, so grow over stripes. B.C, note graded
response to levels of ephrin-A5.
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How are dorsoventral and nasotemporal patterns of
transcription factor set up in the developing
retina?
Extrinsic factors retinoic acid has been shown
to ventralise the optic cup (Ross et al. (2000)
Physiol. Rev. 80, 1021-1054).
Intrinsic factors the prior expression of other
genes e.g Bmp4 dorsally /Ventropin ventrally
(antagonists) Pax6 (Pax6-/- mice - the optic
vesicle does not express any dorsal or
nasotemporal markers). Nicole Baumer et al.
(2002) Pax6 is required for establishing the
naso-temporal and dorsal characteristics of the
optic vesicle. Development 129, 4535-4545.
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Dorso-ventral and naso-temporal specification of
the retina
Regionally restricted patterns of expression of
transcription factors imposes dorso-ventral and
naso-temporal specificity in cells within the
developing optic cup.
These transcription factors, directly or
indirectly, control the expression of different
cell surface molecules in developing retinal
ganglion cells from different parts of the
retina. (Ephrin receptors)
This causes the axons from retinal ganglion cells
localised in different parts of the retina to
project to different, specific points in the
lateral geniculate nucleus and superior
colliculus.
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Dorso-ventral and naso-temporal specification of
the retina
Vax genes ventralise the embryonic eye.
(Homeodomain transcription factors ) Mui et al.,
2005. Genes Dev. 19, 1249-1259.
Expression of Vax genes in optic stalk represses
Pax6. Loss of Vax genes optic stalk retains
retinal-like, ventral retina not develop.
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Dev. Biol. 251, 59-73. The retina is divided
into multiple D/V domains.
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D
Tbx5 Ephrin B1
foxd2 EphA3
foxg1
N
T
Vax1,2 EphB3
V
Viral misexpression
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Maureen Peters. 2002. Patterning of the neural
retina. Curr. Opin. Neurobiol. 12, 43-48.
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Conservation of genetic control of eye development
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Concept of homology. things being the same
because they are evolutionarily
related Homologous structures e.g. vertebrate
forelimbs look different but are all derived
from the front legs of a common ancestor.
time
Acanthostega
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Concept of homology. things being the same
because they are evolutionarily related Same
principle applies for homologous genes.
Gene X Gene Y
Gene X Gene Y
Gene X Gene Y
time
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Pax6 is required for eye development in mice and
flies
Pax6/
Pax6/
Pax6/-
Pax6-/-
Pax6-/-
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Concept of homology. so is the development of
homologous structures controlled by homologous
genes? can the eyes of invertebrates and
vertebrates be homologous?
Pax6
Pax6
Pax6
time
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It requires little persuasion to be convinced
that the lens eye of a vertebrate and the
compound eye of an insect are independent
evolutionary events. Ernst Mayr, 1961. (but.
?rhodopsin?).
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Fly eye development is very different from
vertebrate. Imaginal discs small epithelial
sheets tucked away towards the front of the
larva.
When the larva metamorphoses, imaginal discs
proliferate and grow and develop into the adult
structures. Separate imaginal discs for labia
(jaws), eye/antennae, legs, wings, halteres,
genitals.
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Fly eye development is very different from
vertebrate. Eye imaginal disc fused with
antennal imaginal disc.
A photoreceptor cell (rhombomere 8) is specified
in sheet of pluripotent cells - recruits all the
other photoreceptor and support cells. This
happens independently for all ommatidia.
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Pax6 has been called the master regulatory gene
for eye development.
Required in many tissues throughout eye
development Loss of function leads to loss of
eyes in mice and flies. Expression is conserved
in eyes in many different phyla with many
different designs of eye, incl. octopus, clams,
photosensitive ocellus of Ascidians, flatworms.
Ectopic expression in leg/wing/antennae imaginal
discs of Drosophila leads to formation of ectopic
eyes (Pax6 sufficient to override the genetic
programming of imaginal discs and make them form
functional eyes).
Halder et al., Science 1995
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But, lots of genes can create ectopic eyes in
Drosophila.
Organised into 4 families
PAX6 family Members are eyeless ( Pax6), twin of
eyeless, eyegone. DNA-binding transcription
factors. EYA (eyes absent) Has protein-binding
domains that interact with members of the
following two families. SIX family sine oculis,
Optix Have DNA binding and protein-binding
domains. Binds eya to form functional
transcription factor. DACH (dachshund) Shown to
interact with eya. May be transcriptional
cofactor.
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Cross-regulation and autoregulatory loops
involving the members of the master genes might
explain why fly eye development fails if any one
of the families is missing.
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Members of the PAX6, EYA, SIX and DACH families
all show some characteristics of master
regulators of Drosophila eye development. i.e.
Loss of function of any of the genes leads to
loss of eyes. Ectopic expression in leg or wing
primordia leads to formation of ectopic eyes.
Vertebrates have homologues of all these
families, many of which are expressed in the
eyes. Gene duplications have occurred. PAX6
Pax6. EYA Eya1-4, Eya2, Eya3,
Eya4. SIX Six1-6 DACH Dach1, Dach2.
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A conserved team at the centre of eye development?
Hanson, I. M. (2001) sem. in Cell Dev. Biol.
12, 475-484.
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PAX6, SIX, EYA and DACH genes in vertebrate eye
development
PAX6 - Expressed throughout eye
development. Loss of function leads to failure
of eye development. Ectopic expression leads to
ectopic eye structures. EYA - Eya1, Eya2, Eya3
expressed in eye development. Human EYA1-/-
leads to ocular abnormalities. SIX - Six3,
Six4, Six5, Six6 expressed in eyes Six3
expression is dependent on Pax6 in lens, but not
retina. Six3-/- leads to eye abnormalities Ectop
ic expression of Six3 and/or Six6 in brain leads
to ectopic expression of retinal
markers/structures. Six3 and Six6 both
important for determination of retinal cell
fates. DACH- Dach1, Dach2 expressed in eyes.
Function unknown.
DETAILS NOT IMPORTANT. JUST REMEMBER THAT THE
GENES THATARE IMPORTANT FOR EYE DEVELOPMENT IN
DROSOPHILA ARE OFTEN IMPORTANT FOR EYE
DEVELOPMENT IN VERTEBRATES TOO!
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PAX6, SIX, EYA and DACH genes in vertebrate eye
development
Look carefully at gene expression and
interactions things not THAT simple. Purcell et
al., 2005. Gene Expression Patterns

Sequence and general ocular expression of e.g.
Six3, Eya1, Dach 1, Pax6 is conserved. But not
all genes expressed together in same cells all
the time (note CMZ). The expression of one gene
doesnt always require expression of the
others. Dach1 and Eya1 not absolutely required
for eye development.
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Even in Drosophila not all tissues that
normally express the PAX6, EYA, SIX, DACH genes
go on to form eyes
The PAX6, EYA, SIX, DACH interaction might be a
conserved regulatory network that can drive
differentiation of many tissues, with specificity
depending on other extrinsic or intrinsic signals.
E.g. during vertebrate limb development, Six1,
Eya2, Dach2 and Pax3 are all co-expressed and
together drive myogenesis. Eya-1 and Pax6 genes
interact during development in C. elegans, which
doesnt have eyes (Furuya, M. et al., 2005)
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Expression of Pax2 and Pax6 is non-overlapping in
Drosophila and vertebrate eyes.
Pichaud and Desplan, 2002. Curr. Opin. Genet.
Dev. 12, 430-434.
Drosophila Pax6 in undifferentiated multipotent
cells ahead of morphogenetic furrow. Pax2 in
differentiating support cells. Vertebrate Pax6
in several locations, including undifferentiated,
multipotent retinal progenitor cells. Pax2
in differentiating optic stalk (support cells).
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Prox 1 (remember that?) is the vertebrate
homologue of the Drosophila gene prospero
(Pros) prospero is expressed in the developing
photoreceptors (rhabdomeres) of the Drosophila
eye. Loss of Pros transforms rhabdomere 7 into
rhabdomere 8 I.e. transforms one type of retinal
cell into another just like in vertebrates See
Cook, 2003. BioEssays 25, 921-925
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atonal ( ath5, Math5) and hedgehog ( Shh) in
vertebrate and invertebrate eyes
A Mexican wave of atonal / ath5 and hh / Shh
expression proceeds from optic stalk outwards
through the undifferentiated retina and precedes
the differentiation of the first retinal neurons
(R8 or RGCs). hh atonal neurogenesis
Jarman, A. P. (2000) Curr. Biol. 10, R857-R859.
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Summary Vertebrate and invertebrate eyes use
same or similar genetic pathways during
development. Homologous genes, deployed in
similar areas doing similar jobs. Implies that
these genetic pathways were used to build eyes in
last common ancestor (a disgusting worm-like
thing). BUT. There are important
differences... e.g. vertebrate Rx gene, critical
from earliest stages for formation of optic
vesicle and retina - Drosophila homologue Drx not
expressed in eyes. When examine details of action
of genes, or tissues where genes expressed,
apparent similarities become a lot more
complicated/less convincing.
Reading Jarman, A. P. (2000). Vertebrates and
insects see eye to eye. Curr. Biol. 10,
R857-859. Kumar J. P. and Moses, K. (2001) Eye
specification in Drosophila perspectives and
implications. Sem. In Cell Dev. Biol. 12,
469-474. Peters, M. A. (2002). Patterning the
neural retina. Curr. Opin. Neurobiol. 12, 43-48.
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Possibility 1. All eyes are homologous structures
derived from an ancestral eye in last common
ancestor
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Possibility 2. Last common ancestor didnt have
eyes, but used Pax6/so/eya genes to pattern
anterior CNS. Eyes evolved independently, but
hijacked the same developmental motor.